The goal of this research is to elucidate the critical cellular changes that lead to irreversible cell injury. These mechanisms must be understood before rational interventions can be proposed. Previously, we have shown that an increase in cytosolic free calcium is associated with cell injury. In many systems, it has been observed that exposure of cells or animals to a brief stress provides considerable protection against a subsequent more severe stress. This brief period of stress is known to result in the synthesis of stress proteins; however, in spite of the identification of these proteins, there is little understanding of the mechanisms of protection. Exposing perfused rat heart to several brief (5 minute) periods of anoxic stress (referred to as preconditioning) affords protection when these preconditioned hearts are subjected to a longer 30-60 minute period of anoxic stress such that the preconditioned hearts have less necrosis and better recovery of function compared to untreated controls. Using 31P nuclear magnetic resonance(NMR) we have determined that preconditioning decreased the rate of ATP utilization; preconditioned hearts have a slower rate of decline in ATP and a reduced rate of anaerobic glycolysis. In addition, using a combination of 31P NMR and 19F NMR (to measure intracellular calcium) we have determined that the reduced metabolism results in less H+ production, and therefore less Na/H and Na/Ca exchange and ultimately less of an increase in cytosolic free calcium. We conclude that at least part of the protective effect of preconditioning is due to reduced calcium overload. We were interested in addressing the mechanism(s) responsible for the reduced rate of ATP utilization. We have investigated the hypothesis that adenosine is the mediator of preconditioning; however, in perfused rat heart adenosine does not appear to play such a role since we were unable to block the effect with an adenosine antagonist. In addition, we were not able to block preconditioning by the addition of glibenclamide, thus ruling out a role for the ATP sensitive K channels as part of this process.